铁皮石斛促分裂原活化蛋白激酶基因DoMPK2的分子特征

张岗,张大为,赵明明,郭顺星

中国药学杂志 ›› 2013, Vol. 48 ›› Issue (19) : 1654-1659.

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中国药学杂志 ›› 2013, Vol. 48 ›› Issue (19) : 1654-1659. DOI: 10.11669/cpj.2013.19.013
论著

铁皮石斛促分裂原活化蛋白激酶基因DoMPK2的分子特征

  • 张岗12,张大为2,赵明明2,郭顺星2
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Molecular Characterization of A Mitogen-Activated Protein Kinase Gene DoMPK2 in Dendrobium officinale

  • ZHANG Gang1,2, ZHANG Da-wei2, ZHAO Ming-ming2, GUO Shun-xing2
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摘要

目的克隆铁皮石斛(Dendrobiumofficinale)促分裂原活化蛋白激酶(mitogen-activatedproteinkinase,MAPK)基因DoMPK2并进行分子特征分析。方法采用反转录聚合酶链式反应(RT-PCR)、cDNA末端快速扩增(RACE)技术分离全长基因;生物信息学分析编码蛋白的理化特性、保守结构域等特征;用DNASTAR、MEGA软件分别进行氨基酸多序列比对和进化树构建分析;借助实时定量PCR技术检测基因组织表达模式。结果DoMPK2基因cDNA全长1585bp(GenBank注册号JX297595),编码一条由369个氨基酸组成的肽链,相对分子质量42120,等电点6。56。DoMPK2蛋白包含MAPK家族保守的丝氨酸/苏氨酸蛋白激酶催化结构域(32~-319)和MAP激酶的保守位点(67~170)。DoMPK2与多种植物MAPK基因高度相似(56%~84%),与水稻、玉米等单子叶植物MAPK基因亲缘关系较近。DoMPK2为组成型表达,其转录本在石斛种子和根中的相对表达量较高,分别为叶中的9。696、2。327倍,茎和叶中的表达变化不显著。结论DoMPK2基因的分子特征为进一步研究其在铁皮石斛生长发育、抗逆胁迫等生命活动过程中的生物学功能奠定基础。

Abstract

OBJECTIVE To clone and characterize a mitogen-activated protein kinase (MAPK) gene DoMPK2 in Dendrobium officinale. METHODS RT-PCR and RACE technologies were used for gene clone. Characteristics including the physicochemical properties and conserved domain of the deduced DoMPK2 protein were determined using a series of bioinformatics tools. The analyses of multiple alignment and phylogenetic tree were performed using DNASTAR and MEGA softwares, respectively. qPCR was employed to examine the tissue specific expression pattern of DoMPK2. RESULTS The full length cDNA of DoMPK2 was 1 585 bp (GenBank accession No. JX297595) and encoded a 369-aa protein with a molecular weight of 42.12 kD and an isoelectric point (pI) of 6.54. The deduced DoMPK2 protein contained the conserved serine/threonine- protein kinase catalytic domain (32-319) and MAP kinase signature (67-170). Multiple sequence alignment and phylogenetic analysis demonstrated that DoMPK2 was highly similar (56%-84%) to MAPK genes from various plants and was closely related to rice and maize monocots. Real time quantitative PCR (qPCR) analysis revealed that DoMPK2 was constitutively expressed in the four included tissues. The transcripts were the most abundant in the seeds and roots, which were 9.696 and 2.327 fold over the leaves, and there had no significant expression in the stem and leaf samples. CONCLUSION Molecular characterization of DoMPK2 will be useful for further elucidation of the functions of this gene involving in the plant growth and development and stress response of D. officinale.

关键词

铁皮石斛 / 激酶 / 克隆 / 基因表达 / 实时定量PCR

Key words

Dendrobium officinale / kinase / cloning / gene expression / real time quantitative PCR

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张岗,张大为,赵明明,郭顺星. 铁皮石斛促分裂原活化蛋白激酶基因DoMPK2的分子特征[J]. 中国药学杂志, 2013, 48(19): 1654-1659 https://doi.org/10.11669/cpj.2013.19.013
ZHANG Gang,, ZHANG Da-wei, ZHAO Ming-ming, GUO Shun-xing. Molecular Characterization of A Mitogen-Activated Protein Kinase Gene DoMPK2 in Dendrobium officinale[J]. Chinese Pharmaceutical Journal, 2013, 48(19): 1654-1659 https://doi.org/10.11669/cpj.2013.19.013
中图分类号: R97   

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基金

国家自然科学基金资助项目(31070300; 31101608);陕西省青年科技新星计划项目(2012KJXX-44);陕西省教育厅专项科研计划项目(2013JK0829)
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